DNA polymerases synthesize DNA molecules in the 5′ to 3′ direction from deoxynucleoside triphosphates (nucleotides) using a complementary template DNA strand and a primer by successively adding nucleotides to the free 3′-hydroxyl group of the growing strand. The template strand determines the order of addition of nucleotides via Watson-Crick base pairing. In cells, DNA polymerases are involved in DNA repair synthesis and replication. See, e.g., Kornberg et al., 1992, DNA Synthesis, W. H. Freeman, New York; Alberts et al., 1994, Molecular Biology of the Cell, 3d ed., Garland Press, New York.
Many molecular cloning techniques and protocols involve the synthesis of DNA in in vitro reactions catalyzed by DNA polymerases. For example, DNA polymerases are used in DNA labelling and DNA sequencing reactions, using either 35S-, 32P-, 33P- or fluorescently-labelled nucleotides. One of the most versatile and widely-used DNA synthesis techniques, the polymerase chain reaction (PCR) technique, is disclosed in U.S. Pat. Nos. 4,683,202; 4,683,195; 4,800,159; and 4,965,188, and discussed in PCR Strategies, 1995 Innis et al. (eds.), Academic Press, Inc., incorporated herein by reference in their entireties.
The best characterized DNA polymerase is Escherichia coli DNA polymerase I (Eco Pol I). Eco Pol I and several DNA polymerases homologous to it have three enzymatic functions: i) a 5′-3′ exonuclease activity, ii) a 3′-5′ exonuclease activity and iii) a DNA synthesis activity. The latter two functions are located toward the carboxy terminus of the protein, within a ‘Klenow’ fragment (Eco Pol I K). Enzyme preparations of Eco Pol I can be treated with subtilisin to yield a Eco Pol I K minus the 5′-3′ exonuclease activity. See Brown et al., 1982, J Biol. Chem. 257: 1965–72; Joyce et al., 1982, J. Biol. Chem. 257: 1958–64; Joyce et al., 1983, Proc Natl Acad Sci USA 80: 1830–34; Klenow et al., 1970, Proc. Natl. Acad. Sci. USA 65: 168–75; Kornberg, 1974; Setlow, P. et al., 1972, J. Biol. Chem. 247: 224–31; Setlow et al., 1972, J. Biol. Chem. 247: 232–40; Steitz et al., 1987, in Protein Engineering, Chap. 20, pp. 227–35 Oxender et al. (eds). Alan R. Liss, New York.
Crystal structure analysis of Eco Pol I K has shown that its peptide chain is folded into two distinct domains, with the smaller domain, of 200 amino acid residues, being the 3′-5′ exonuclease domain and the other domain, of 400 amino acid residues, being the DNA synthesis domain. See Ollis et al., 1985, Nature 313:762–66. The active sites of the 3′-5′ exonuclease and DNA synthesis activities are separated by about 30 angstroms. See id. The DNA synthesis active site binds to double-stranded DNA containing a single-stranded 5′ extension and deoxynucleoside triphosphate (dNTP) whereas the 3′-5′ exonuclease active site binds to single-stranded DNA and deoxynucleoside monophosphate (dNMP). See id. The existence of a conserved 3′-5′ exonuclease active site present in a number of DNA polymerases was predicted by Bernat et al., 1989, Cell 59: 219–28; Blanco et al., 1992, Gene 112: 139–44; Reha-Krantz, 1992, Gene 112: 133–37.
The DNA synthesis domain of Eco Pol I has been cloned, expressed, and characterized independently of the 3′-5′ and 5′-3′ exonuclease domains. As discussed above, the DNA synthesis domain contains approximately 400 amino acids. There is 50-fold less polymerase activity in the DNA synthesis domain than in the Eco Pol I K. See Derbyshire et al., 1993, Nucleic Acids Res. 21:5439–48.
Thermostable and thermoactive DNA polymerases derived from a variety of organisms have been described extensively in the literature. Particular examples include DNA polymerases from a variety of species of the eubacterial genus Thermus, see U.S. Pat. No. 5,466,591, in particular from Thermus aquaticus (Taq DNA polymerase) described in U.S. Pat. Nos. 4,889,818, 5,352,600 and 5,079,352, and the DNA polymerase from the eubacterial species Thermotoga maritima (Tma DNA polymerase) described in U.S. Pat. Nos. 5,374,553 and 5,420,029.
Both E. coli, a mesophile, and T. maritima, a thermophile, are eubacteria. Like E. coli DNA polymerase I, Tma DNA polymerase has both a 5′-3′ exonuclease activity and a 3′-5′ exonuclease activity. In contrast, DNA polymerases from Thermus species, which also are thermophilic eubacteria, possess only 5′-3′ exonuclease activity. A review of thermostable and thermoactive DNA polymerases and their associated activities is found in Abramson, 1995, in PCR Strategies, Innis et al. (eds), Academic Press, Inc. Mutant forms of a number of archae and eubacterial thermostable or thermoactive DNA polymerases that lack a 3′-5′ exonuclease activity are described in U.S. Pat. Nos. 6,015,668, 5,939,301, 5,988,614, 5,882,904, 5,489,523.
While Eco Pol I and Tma DNA polymerase have the same three enzymatic activities, and the same general domain structure, they are very different enzymes. For example, Eco Pol I and Tma DNA polymerase have very different amino acid sequences. In particular, even when gaps are introduced into their sequences to optimize their alignment, the 3′-5′ exonuclease domains of these two proteins are only about 33% identical. Further, the two enzymes exhibit different specific activities and differing resistance to chemical denaturing conditions. As these differences are observed in purified enzymes removed from their natural in vivo environments, they must be caused by differences in the amino acid sequences of the enzymes themselves.
The 3′-5′ exonuclease activity “proofreads” the nascent DNA strand as it is synthesized and preferentially removes nucleotides from it that are mismatched with the template strand, thus increasing the fidelity of DNA synthesis. However, the presence of a robust 3′-5′ exonuclease activity can be problematic for in vitro polymerization reactions, particularly PCR, in which the presence of a 3′-5′ exonuclease activity lowers the efficiency of the amplification. See Barnes, 1994, Proc. Natl. Acad. Sci. USA 91:2216–20.
Attempts have been made to avoid the deleterious effects of 3′-5′ exonuclease activity by using a DNA polymerase enzyme that lacks a 3′-5′ exonuclease activity. Taq polymerase naturally lacks this activity. Other DNA polymerases have been genetically engineered to eliminate it. See, e.g., U.S. Pat. Nos. 6,015,668, 5,939,301, 5,988,614, 5,882,904 and 5,489,523. These enzymes work in applications where higher fidelity of replication is unnecessary, for example, in a PCR used to amplify a template for DNA sequencing reactions, size analysis, restriction enzyme analysis or probe-based analyses.
However, other polymerization reactions, e.g., PCR to amplify a DNA fragment for cloning, require a higher level of fidelity. In an effort to reduce the problems associated with the 3′-5′ exonuclease activity while retaining its benefits, mixtures of two different DNA polymerases have been used. One polymerase in the mix has a wild-type level of 3′-5′ exonuclease activity. The other lacks this activity. The ratio of the two polymerases is manipulated to produce the desired ratio of 3′-5′ exonuclease to DNA polymerase activities. See Cheng et al., 1994, Proc. Natl. Acad. Sci. USA 91:5695–99; Barnes, 1994, Proc. Natl. Acad. Sci. USA 91:2216–20; Cheng, 1995, “Longer PCR Amplifications” in PCR Strategies (Innis et al., eds), Academic Press, San Diego 313–24; Cheng et al., 1995, PCR Meth. Applic. 4:294–98; U.S. Pat. Nos. 5,512,462, 5,436,149 and 5,556,772 and PCT Pat. App. Pub. WO 94/26766. While this blending technique can be used to achieve the desired amount of 3′-5′ exonuclease activity, it is expensive, time consuming, and must be optimized on a batch-by-batch basis. It requires the DNA polymerase activities and 3′-5′ exonuclease activities of each enzyme preparation to be carefully calibrated so that the two polymerases can be mixed in the right amounts to produce the desired ratio of 3′-5′ exonuclease activity to DNA polymerase activity. Thus, there is a need in the art for a rapid and economical method of producing a DNA polymerase reagent with a desired level of 3′-5′ exonuclease activity and a desired ratio of 3′-5′ exonuclease to DNA polymerase activities.